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Recombineering
Recombineering (recombination-mediated genetic engineering)〔Ellis, H. M., D. Yu, T. DiTizio & D. L. Court, (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc. Natl. Acad. Sci. USA 98: 6742-6746.〕 is a genetic and molecular biology technique based on homologous recombination systems, as opposed to the older/more common method of using restriction enzymes and ligases to combine DNA sequences in a specified order. Recombineering is widely used for bacterial genetics, in the generation of target vectors for making a conditional mouse knockout, and for modifying DNA of any source often contained on a bacterial artificial chromosome (BAC), among other applications.
==Development==
Although developed in bacteria, much of the inspiration for recombineering techniques came from methods first developed in Saccharomyces cerevisiae 〔Orr-Weaver, T. L., J. W. Szostak, et al. (1983) Genetic applications of yeast transformation with linear and gapped plasmids. Methods. Enzymol. 101: 228-245.〕 where a linear plasmid was used to target genes or clone genes off the chromosome. In addition, recombination with single-strand oligonucleotides (oligos) was first shown in Saccharomyces cerevisiae.〔Moerschell, R. P., S. Tsunasawa, et al. (1988). "Transformation of yeast with synthetic oligonucleotides." Proceedings of the National Academy of Sciences of the United States of America 85(2): 524-528.〕 They saw recombination with oligonucleotides as short as 20 bases.
Recombineering is based on homologous recombination in ''Escherichia coli'' mediated by bacteriophage proteins, either RecE/RecT from Rac prophage 〔Zhang, Y., F. Buchholz, J. P. Muyrers & A. F. Stewart, (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nature Genetics 20: 123-128.〕 or Redαβδ from bacteriophage lambda.〔Muyrers, J. P., Y. Zhang, G. Testa & A. F. Stewart, (1999) Rapid modification of bacterial artificial chromosomes by ET- recombination. Nucleic Acids Res. 27: 1555-1557.〕〔Yu, D., H. M. Ellis, et al. (2000). "An efficient recombination system for chromosome engineering in Escherichia coli." Proceedings of the National Academy of Sciences of the United States of America 97(11): 5978-5983.〕 The lambda Red recombination system is now most commonly used and the first demonstrations of Red ''in vivo'' genetic engineering were independently made by Kenan Murphy〔Murphy, K. C., (1998) Use of bacteriophage λ recombination functions to promote gene replacement in Escherichia coli. J. Bacteriol. 180: 2063-2071.
〕 and Francis Stewart.〔〔 However, Murphy's experiments required expression of RecA and also employed long homology arms. Consequently the implications for a new DNA engineering technology were not obvious. The Stewart lab showed that these homologous recombination systems mediate efficient recombination of linear DNA molecules flanked by homology sequences as short as 30 base pairs (40-50 base pairs are more efficient) into target DNA sequences in the absence of RecA. Now the homology could be provided by oligonucleotides made to order, and standard ''recA'' cloning hosts could be used, greatly expanding the utility of recombineering.
抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』
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